Induction of skeletal muscle differentiation in vitro by therapeutic ultrasound

Therapeutic ultrasound (TU) has been used for the last 50 y in rehabilitation, including treatment of soft tissues. Ultrasound waves can be employed in two different modes of operation, continuous and pulsed, which produce both thermal and non-thermal effects. Despite the large-scale use of TU, there are few scientific studies on its biologic effects during skeletal muscle differentiation. To better analyze the cellular effects of TU, we decided to follow cells in vitro. The main purpose of this study was to evaluate the effects of TU in primary chick myogenic cell cultures using phase contrast optical microscopy and immunofluorescence microscopy, followed by image analysis and quantification. Our results indicate that TU can stimulate the differentiation of skeletal muscle cells in vitro, as measured by the thickness of multinucleated myotubes, the ratio of mononucleated cells to multinucleated cells and expression of the muscle-specific protein desmin. This study is a first step toward a metrologic and science-based protocol for cell treatment under different ultrasound field exposures.